Activation of skeletal muscle fibers, which is a prerequisite for all bodily movement as well as for respiration, is initiated by electrical depolarization of the transverse tubules (TTs), causing membrane voltage (V) sensors in the TT dihydropyridine receptor (DHPR) to trigger Ca2+ release via the abutting skeletal muscle ryanodine receptor (RyR1)/Ca2+ release channels in the adjacent sarcoplasmic reticulum membrane. However, the molecular mechanisms coupling the TT V sensor to SR RyR1 release activation are poorly understood, and the roles of various modulatory molecules, including S100A1 and calmodulin (CaM) are not clear. These issues are important since any pathologic interference with the Ca2+ release activation process may modify or disrupt muscle function. Here in Aim 1 we identify a previously totally unsuspected marked suppression of muscle Ca2+ release in a transgenic mouse model expressing a hypokalemic periodic paralysis (hypoPP) CaV1.1 V sensor charge mutation, and characterize the mechanism(s) underlying this defect in muscle activation. Muscle Ca2+ release is also modulated by a variety of accessory proteins. During the current grant cycle we have made the novel finding that the Ca2+ binding protein S100A1 binds to the previously identified calmodulin (CaM) binding domain (CaMBD) in RyR1, which should now be referred to as a CaM/S100A1 binding domain since these molecules interact for binding at this site. In Aims 2 and 3 we utilize shRNA techniques to suppress the protein expression of S100A1, CaM or both S100A1 and CaM to investigate the effects of each of these ligands as well as their competitive interaction at sites other than the CaMBD of RyR1. We will use high speed (<50 us/line) line-scan confocal imaging of fibers containing the Ca2+ indicator fluo-4 to monitor Ca2+ signals and calculate the underlying Ca2+ release flux from the SR during single or trains of action potentials in intact fibers, or during voltage clamp depolarization of whole cell voltage clamped fibers with high levels of EGTA in the patch pipette solution. We will use adult muscle fibers with molecular biologically manipulated expression of endogenous or exogenous proteins. Parallel NMR and binding studies will examine the structures and binding affinities of S100A1 and/or CaM binding to peptides corresponding to the identified binding sites. This project will elucidate basic molecular mechanisms regulating Ca2+ release in skeletal muscle and the roles of voltage sensor charges that are mutated in hypoPP in muscle Ca2+ release. It will characterize the modulation of SR Ca2+ release by S100A1 and CaM, which might be compromised in generalized or specific muscle disease states, or in aging muscle. Thus, this project has high impact for multiple disciplines, and for problems of both locomotion and breathing common to a variety of advanced diseased states and aging.